1. Evaluating a Blood Film, WBC Morphology, WBC Blood Parasites and
Morphologic Abnormalities of Blood Cells
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2. Manual Determination of WBC
An improved Neubauer hemocytometer is shown on the left, and the counting chamber is shown on the right. 
WBCs are counted in the four large corner squares of the grid, and the number of WBCs is multiplied by 50 to determine WBCs/µL.
The pink circle indicates a corner square, which can be visualized through the 10× objective.  
Illustration by Tim Vojt, Biomedical Media, The Ohio State University College of Veterinary Medicine. Copyright The Ohio State University

3. Preparing a Blood Smear
Microscopic evaluation of a blood smear is an important component of a CBC because some hematologic abnormalities are recognized only on the blood smear.
Preparation of high-quality blood smears is a technical skill that requires practice. Blood smears should be made with new, clean microscope slides using one of several methods.
Most commonly, a small drop of blood is placed close to one end of a microscope slide that is held stationary on a flat surface, and a second “spreader” slide is used to push the drop forward in a smooth, even motion, resulting in a blood smear that is evenly distributed across the stationary slide.
Illustration by Tim Vojt, Biomedical Media, The Ohio State University College of Veterinary Medicine. Copyright The Ohio State University

4. Blood Smears Dry quickly to prevent RBC artifacts
Stain within 48 hours
Store at room temperature
Ship separately from biopsy specimens exposed to formalin
Blood smears should be dried quickly before staining to prevent RBC artifacts.
Slides should be labeled at one end with the date and the animal’s name or identification number and ideally should be stained within several hours. A lapse longer than 48 hours may result in inadequate staining.
Unstained smears being sent to a reference laboratory should be stored at room temperature, not in a refrigerator or freezer because water condensation will damage the cells.
Unstained smears should be stored away from formalin fumes and shipped separately from surgical biopsy specimens that have been placed in formalin because formalin vapors inhibit optimal staining.

5. Blood Smear Evaluation
Three regions
Counting area: between body and feathered edge
Accurate evaluation of blood smears requires a systematic procedure for examination and expertise in identifying normal cells, morphologic abnormalities, and artifacts.
A blood smear consists of three regions: body, counting area, and feathered edge.
Illustration by Tim Vojt, Biomedical Media, The Ohio State University College of Veterinary Medicine. Copyright The Ohio State University

6. Agglutination in Blood Smear
Rouleaux formation
RBCs in chains that look like stacks of coins
Agglutination
Irregular RBC clumps
Excess antibody binding
IHA in dogs
In the body of the smear, RBCs can be evaluated for rouleaux formation or agglutination.
Rouleaux formation refers to RBCs in chains that resemble a stack of coins.
In horses and cats, rouleaux formation is common, whereas in dogs, rouleaux formation may be an indication of inflammation (increased plasma proteins), or it may be an artifact of smear preparation.
Agglutination refers to irregular, variably-sized clumps of RBCs that form because of excess antibodies bound to the surface of RBCs.

7. WBC Differentiation Marked species variation
Differential WBC count: Identify and enumerate at least 100 leukocytes in counting area of blood smear
Percentage of each cell type × total WBC (in mcg/L of blood)
Classifications
Segmented neutrophils, band neutrophils, eosinophils, lymphocytes, monocytes
Determination of the numbers of each type of WBC can be helpful in establishing a list of differential diagnoses.
A differential WBC count is performed by identifying and enumerating a minimum of 100 leukocytes consecutively encountered in the counting area of the blood smear.
At least 200 cells should be counted if the WBC count is increased.

8. Neutrophil Morphology
Segmented neutrophil
Nucleus
Cytoplasmic granules
Heterophils
Band neutrophil
Metamyelocyte
Figure 12-14A shows a segmented neutrophil (arrowhead), three bands (short arrows), and a metamyelocyte (long arrow).
The bands have C- or U-shaped nuclei. The metamyelocyte has an indented nucleus.

9. Neutrophil Morphology
Left shift
Increase in circulating neutrophils
Regenerative
Indicates release of less mature cells to meet demand
Degenerative
Band neutrophils outnumber segmented; poor prognosis
Toxic changes
Intense neutrophil production
Shortened maturation time
Mild, moderate, or marked
Rarely, formation of giant neutrophils occurs.
Toxic changes can be subjectively reported as mild, moderate, or marked, depending on the percentages of cells affected and the severity of the change.

10. Eosinophil Circulate in low numbers in health
Mediate hypersensitivity reactions
Protect against some parasites
All nucleated cells are eosinophils.

11. Eosinophils, Cont'd
Eosinophilia = increased numbers (allergic/defensive reaction to parasites)
Eosinopenia = decreased numbers (response to corticosteroids)
Marked species variation
There are three segmented neutrophils and an eosinophil in this blood smear from a horse.
In horses, eosinophils have numerous large granules, so they are easy to identify.

12. Basophil Morphology Very low numbers in healthy smears
Basophilia = increased numbers (allergic/defensive reaction to parasites)
Morphology varies with species
There is a basophil on the left and an eosinophil above a neutrophil on the right in this blood smear from a dog.
Basophils in dogs have a few dark granules in the cytoplasm.
Basophils appear larger than neutrophils, and the nuclei appear twisted with chromatin that is slightly less condensed than in neutrophil and eosinophil nuclei.

13. Lymphocytes Predominant circulating cell in cattle, sheep, goats
Usually smaller than neutrophils
Round nucleus almost fills cytoplasm
Lymphocytosis
Antigenic stimulation
Lymphoid neoplasia
Acute lymphoid leukemia (ALL)
Chronic lymphocytic leukemia (CLL)
Figure 12-14G (top) shows a normal lymphocyte in upper portion and a reactive lymphocyte in the lower portion of blood smear from a cat.
Figure 12-14I (bottom) shows neoplastic lymphocytes in ALL in a dog.

14. Monocytes Relatively low numbers Similar in most species
Larger than neutrophils
Abundant gray-blue cytoplasm
Monocytosis: inflammataion or hemolysis
Reactive: chemotherapy or inflammation
Figure 12-14K shows two monocytes in equine blood smear.
Shows typical abundant basophilic cytoplasm with clear vacuoles.
Nuclei are indented or irregular.

15. WBC Blood Parasites

16. Ehrlichia canis
Disease in dogs
Vector: Rhipicephalus sanguineus, Dermacenter variabilis.
Cells: Monocytes, macrophages, lymphocytes. Morulae rarely seen in infected dogs and usually only in acute infection (Allison and Little 2013).
Clinical signs: Fever, lethargy, anorexia, excess hemorrhage (attributed to platelet dysfunction as well as thrombocytopenia – thrombocytopenia is usually not severe enough to induce hemorrhage alone – platelet counts are usually moderately decreased, i.e ,000/uL). Some dogs have neurologic disease and may have increased numbers of granular lymphocytes in their cerebrospinal fluid (this finding is not specific for this organism).

17. Anaplasma platys previously knowns as Ehrlichia platys
Two main species of Anaplasma are thought to infect dogs – Anaplasma phagocytophilum (aka Ehrlichia equi) and Anaplasma platys. The former infects granulocytes (neutrophils and eosinophils) whereas the latter infects platelets.

18. Morphologic Changes
In addition to enumerating each type of white blood cell (WBC) and estimating the platelet count, the differential blood count requires that the morphologic features of the cells be evaluated. The presence of any abnormal cells or toxic changes should be semiquantified
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19. Quantifying Morphologic Changes
Two methods
Scale of 1+, 2+, 3+, 4+ to indicated relative percentages of cells with changes
1+ = 5% to 10%
2+ = 10% to 25%
3+ = approximately 50%
4+ = more than 75%
Slight, moderate, marked
Slight = 10%
Moderate = 25%
Marked = 50%
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20. Morphologic Abnormalities in WBCs
Nuclear hyposegmentation
Pelger-Huet anomaly
Congenital defect
Hyposegmentation of all granulocyte nuclei
Nuclear hyposegmentation in neutrophils (N) and eosinophils (E) from a dog with Pelger-Huet anomaly (Wright stain)
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21. Morphologic Abnormalities in WBCs (cont.)
Nuclear hypersegmentation
Very common
Nuclei with 5 or more lobes
Can be due to aging neutrophils either in vivo or in vitro (prolonged storage)
Common in poodles with macrocytosis
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22. Morphologic Abnormalities in WBCs (cont.)
Toxic change
Common disease-induced cytoplasm changes in neutrophils
Associated with inflammation, infection, drug toxicity
Dogs – bacterial infection
Cats – common in not severely ill cats
Cytoplasmic basophilia, Döhle bodies, toxic granulation, giantism
Caused by decreased length of time in the neutrophil maturation process with bone marrow
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23. Toxic Neutrophil Which neutrophil is toxic in this photo?
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24. Intracytoplasmic Inclusions
Common inclusion
Histoplasma capsulatum
Francisella philomiragia
Mycobacterium
Gametocytes of Hepatozoon canis
Amastigotes of Leishmania infantum
Ehrlichia morulae
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25. Atypical Lymphocytes
Atypical lymphocytes have basophilic cytoplasm and cleave nuclei
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26. Reactive Lymphocytes a.k.a. immunocytes Increase basophilic cytoplasm
More abundant cytoplasm
Sometimes a large, convoluted nuclei
Usually caused by antigenic stimulation
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27. Lysosomal Storage Disorders
Rare inherited diseases
A substance abnormally stored in cells
Clinical signs vary
Most skeletal or neurologic disease
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28. Birman Cat Neutrophil Granulation Anomaly
Neutrophils contain fine eosinophilic to magenta granules
Inherited autosomal-recessive trait
Neutrophil function is normal and cats are healthy
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29. Chédiak-Higashi Syndrome
Neutrophils have large, fused lysosomes with the cytoplasm
Stain lightly pink to eosinophilic
Approximately 1 in 3 or 4 neutrophils are affected
Affected animals may bleed due to abnormal platelet function
Normal neutrophil function and animals appear healthy
Persian cats, cattle, foxes, and others
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30. Chédiak-Higashi Syndrome (cont.)
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31. Siderotic Granules
Present in neutrophils and monocytes of animals with hemolytic anemia
Appear as Döhle bodies
Differentiate with Prussian blue stain
Can occur in RBCs (siderocytes)
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32. Smudge Cells a.k.a. basket cells
Degenerative leukocytes that have ruptured
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33. Smudge Cells (cont.)
Karyolysis – degenerative change to the nucleus by dissolution of the nuclear membrane
Septic exudates
Pyknosis – condensing of the nucleus as the cell dies
Karyorrhexis – fragmentation of the nucleus after cell death
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